SummaryStreptococcus sanguis is the most common oral bacterium causing infective endocarditis and its ability to adhere to platelets, leading to their activation and aggregation, is thought to be an important virulent factor. Previous work has shown that S. sanguis can bind directly to platelet glycoprotein (GP) Ib but the nature of the adhesin was unknown. Here, we have shown that a high molecular weight glycoprotein of S. sanguis mediates adhesion to glycocalacin. The bacterial glycoprotein was purified from cell extracts by chromatography on GPIb-and wheatgerm agglutinin affinity matrices and its interaction with GPIb was shown to be sialic acid-dependent. We designated the glycoprotein serine-rich protein A (SrpA). An insertional inactivation mutant lacking the SrpA of S. sanguis showed significantly reduced binding to glycocalacin, reduced adherence to platelets and a prolonged lag time to platelet aggregation. In addition, under flow conditions, platelets rolled and subsequently adhered on films of wild-type S. sanguis cells at low shear (50/s) but did not bind to films of the SrpA mutant. Platelets did not bind to wild-type bacterial cells at high shear (1500/s). These findings help to understand the mechanisms by which the organism might colonize platelet-fibrin vegetations.

Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Objective-Staphylococcus aureus is the most frequent causative organism of infective endocarditis (IE) and is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Previously, we showed that S aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) can stimulate rapid platelet aggregation. Methods and Results-In this study we investigate their relative roles in mediating aggregate formation under physiological shear conditions. Platelets failed to interact with immobilized wild-type S aureus (Newman) at shear rates Ͻ500 s Ϫ1 but rapidly formed an aggregate at shear rates Ͼ800 s Ϫ1. Inactivation of the ClfA gene eliminated aggregate formation at any shear rate. Using surrogate hosts that do not interact with platelets bacteria overexpressing ClfA supported rapid aggregate formation under high shear with a similar profile to Newman whereas bacteria overexpressing FnBPA did not. Fibrinogen binding to ClfA was found to be essential for aggregate formation although fibrinogencoated surfaces only allowed single-platelets to adhere under all shear conditions. Blockade of the platelet immunoglobulin receptor Fc␥RIIa inhibited aggregate formation. Conclusions

Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein's activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.

We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip. iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates. By separating platelets from whole blood using specific binding to protein spots of a defined size, iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (1) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet-platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopulations from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated. To date, we have demonstrated 1-4 of the above list. Platelets were separated from whole blood using iPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 μm). The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state. Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets. For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (αIIbβ3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function. A critical function of platelets is to adhere to regions of damage on blood vessel walls; in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix interactions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

We report a novel device to analyze cell-surface interactions under controlled fluid-shear conditions on well-characterised protein surfaces. Its performance is demonstrated by studying platelets interacting with immobilised von Willebrand Factor at arterial vascular shear rates using just 200 μL of whole human blood per assay. The device's parallel-plate flow chamber, with 0.1 mm² cross sectional area and height-to-width ratio of 1:40, provides uniform, well-defined shear rates along the chip surface with negligible vertical wall effects on the fluid flow profile while minimizing sample volumetric flow. A coating process was demonstrated by ellipsometry, atomic force microscopy, and fluorescent immunostaining to provide reproducible, homogeneous, uniform protein layers over the 0.7 cm² cell-surface interaction area. Customized image processing quantifies dynamic cellular surface coverage vs. time throughout the whole-blood-flow assay for a given drug treatment or disease state. This device can track the dose response of anti-platelet drugs, is suitable for point-of-care diagnostics, and is designed for adaptation to mass manufacture.

Key Points• Wnt signaling is essential for MK proliferation and maturation in addition to profoundly stimulating proplatelet formation.• These observations suggest that mature megakaryocytes may be able to respond to known Wnt gradients in the osteoblastic and vascular niches. IntroductionThe Wingless (Wnt) family of secreted glycoproteins regulates diverse biologic processes, from embryonic development through to aspects of adult homeostasis and disease. The Wnt signaling pathways comprise 19 distinct Wnt ligands, a host of extracellular Wnt-modulating proteins and at least 10 members of the 7 transmembrane domain-containing Frizzled (FZD) receptor family. 1 After ligand-receptor binding, multiple pathways and downstream events are triggered in a context specific manner. The most studied Wnt signaling pathway, the canonical pathway, is mediated by the stabilization and nuclear translocation of ␤-catenin (see Figure 1A). Under unstimulated conditions, ␤-catenin is phosphorylated by casein kinase 1 (CK1) and glycogen synthase kinase-3␤ (GSK3␤) in the axin/APC destruction complex, which tags ␤-catenin for ubiquitination and degradation. Stimulation with a canonical Wnt ligand (such as Wnt3a) inhibits this process, stabilizing cytoplasmic ␤-catenin which then migrates to the nucleus. There, it acts as a transcriptional coactivator in complex with transcription factors, such as T-cell factor/lymphoid enhancer factor (TCF/LEF) family members to regulate gene expression. The canonical Wnt pathway inhibitor dickkopf-1 (DKK1) binds to low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and prevents signal transduction from occurring. Multiple "noncanonical" Wnt signaling pathways also exist. 2 Although these pathways all function in a ␤-catenin independent manner, there is an interplay between canonical and noncanonical signaling in some contexts. 3,4 Recently, Wnt signaling has been implicated in the development 5-7 and function of mature blood cells. 8,9 These have focused predominantly on the regulation of hematopoietic stem cell (HSC) function and T-cell development. Other effects of Wnt signaling on myeloid differentiation have been observed, yet its contribution to megakaryopoiesis and platelet formation remains unexplored.We previously identified a role for the canonical Wnt signaling pathway in the regulation of platelet function, 10 with Wnt3a modulating platelet adhesion and suppressing platelet activation to The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. 14,15 where they extend cytoplasmic protrusions known as proplatelets into sinusoidal blood vessels, from which platelets are ultimately released in the circulation. [16][17][18][19] As several Wnts are expressed in hematopoietic niches, 7 MKs probably encounter an array of Wnt ligands during this maturation process.Her...

Objective-The vascular endothelium constitutes a highly effective fluid/solute barrier through the regulated apposition of intercellular tight junction complexes. Because endothelium-mediated functions and pathology are driven by hemodynamic forces (cyclic strain and shear stress), we hypothesized a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We, therefore, examined the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components, occludin and ZO-1. Methods and Results-For these studies, bovine aortic endothelial cells were subjected to physiological levels of equibiaxial cyclic strain (5% strain, 60 cycles/min, 24 hours). In response to strain, both occludin and ZO-1 protein expression increased by 2.3Ϯ0.1-fold and 2.0Ϯ0.3-fold, respectively, concomitant with a strain-dependent increase in occludin (but not ZO-1) mRNA levels. These changes were accompanied by reduced occludin tyrosine phosphorylation (75.7Ϯ8%) and increased ZO-1 serine/threonine phosphorylation (51.7Ϯ9% and 82.7Ϯ25%, respectively), modifications that could be completely blocked with tyrosine phosphatase and protein kinase C inhibitors (dephostatin and rottlerin, respectively). In addition, there was a significant strain-dependent increase in endothelial occludin/ZO-1 association (2.0Ϯ0.1-fold) in parallel with increased localization of both occludin and ZO-1 to the cell-cell border. These events could be completely blocked by dephostatin and rottlerin, and they correlated with a strain-dependent reduction in transendothelial permeability to FITC-dextran. Conclusions-Overall, these findings indicate that cyclic strain modulates both the expression and phosphorylation state of occludin and ZO-1 in vascular endothelial cells, with putative consequences for endothelial tight junction assembly and barrier integrity. Key Words: occludin Ⅲ ZO-1 Ⅲ endothelium Ⅲ cyclic strain Ⅲ permeability T he vascular endothelial monolayer or endothelium is a dynamic cellular interface between the vessel wall and bloodstream. In addition to regulating the physiological effects of humoral and mechanical stimuli on blood vessel tone and remodeling, the endothelium participates in immune and inflammatory reactions and presents a nonthrombogenic surface for blood flow. 1,2 Moreover, the vascular endothelium constitutes a highly effective barrier, which regulates fluid and solute balance in addition to movement of molecular/ cellular components between bloodstream and tissues. [3][4][5] As such, regulation of endothelial barrier integrity (or permeability) is crucial for vascular homeostasis and is a central pathophysiologic mechanism of many vascular processes, including wound healing, angiogenesis, and vascular diseases. [5][6][7] Barrier function is maintained by the regulated apposition of tight junction and adherens protein complexes between adjacent endothelial cells. The organization of these protein complexes is controlled by a number of physiolog...